DESCRIPTION: The aim of this proposal is to produce a cost-effective kit that will facilitate the production of mouse embryonic stem (ES) cell / blastocyst chimeras to make genetically engineered mice. The kit will be designed so that it can be used by investigators with only limited experience with mouse embryo culture and embryo transfer. Costs of making knock-out mice will be significantly reduced by eliminating the need for local animal colonies for the production of 8-cell mouse embryos by supplying them from a central stock of frozen embryos. The thawed 8-cell embryos will be aggregated with the ES cells and then transferred to the uteri of pseudopregnant surrogate mothers. In Phase II of this application, the following research will be done to produce an optimized kit: (1) a method will be developed to assay rapidly the aggregation of ES cells with 8-cell embryos; (2) KSOM/AA, a new medium that is very effective for the culture of mouse preimplantation embryos, will be optimized for the aggregation of ES cells and mouse embryos; (3) the culture of ES cells on a feeder layer will be improved by replacing the feeder layer with a plate of supporting killed primary mouse embryonic fibroblasts; (4) KSOM/AA will be modified to replace Dulbecco's MEM for the culture of ES cells; and (5) data will be collected to determine which of two inbred strains of mouse (C57BL/6 or FVB/N) are preferable as donors of the 8-cell embryos used for aggregation. PROPOSED COMMERCIAL APPLICATION: NOT AVAILABLE